1. Field of the Invention
The present invention relates to the use of ceruloplasmin (and/or derivatives thereof) in a neuroprotective composition. Preferably, the composition of the invention comprises ceruloplasmin and a combination of pyruvate, antioxidant, lipid(s) such as fatty acids since these four components were found to synergistically protect neurons against oxidative stress.
2. Description of the Prior Art
Reactive oxygen species (ROS) have been implicated in the development of many heart and brain dysfunctions. Ischemia/reperfusion insults to these organs are among the leading causes of mortality in America. These insults are caused by complete or partial local occlusions of heart and brain vasculature, by heart stroke or attack, and by cerebral attacks and trauma to the brain. In addition, ROS are involved in artherosclerotic lesions, in the evolution of various neurodegenerative diseases, and are also produced in association to epileptic episodes, in inflammation, in the mechanisms of action of various neurotoxicants, or as side-effects of drugs.
Until now, no ideal therapeutic agent was known to protect neuronal cells against oxidant species associated with various types of oxidative stress. It would therefore be highly desirable to have such neuroprotective agent.
TRIAD is combination of pyruvate, antioxidant and fatty acids. This composition has been patented in 1997 in the U.S. as a therapeutic wound healing compositions (U.S. Pat. No. 5,652,274). Many related U.S. patents have also been issued for covering the uses of TRIAD in antikeratolytic compositions (U.S. Pat. No. 5,641,814); in anti-fungal compositions (U.S. Pat. No. 5,663,208); in acne healing compositions (U.S. Pat. No. 5,646,190); in anti-inflammatory compositions (U.S. Pat. No. 5,648,380); in dermatological compositions (U.S. Pat. No. 5,602,183); in sunscreen compositions (U.S. Pat. No. 5,674,912); in antihistamine compositions (U.S. Pat. No. 5,614,561); in cytoprotective compositions (U.S. Pat. No. 5,633,285); in wound healing composition affixed to razor cartridges (U.S. Pat. No. 5,682,302); and in regenerating compositions (EP 0 573 465 B1). However, none of these patents discloses or suggests the use of TRIAD as neuroprotective agent.
Ceruloplasmin (CP), is a multifunctional blue-copper plasma protein which has important antioxidant and free radical scavenging properties as well as a ferroxidase I activity. Ceruloplasmin was also shown as an important oxygen free radical (OFR) scavenger. Recent studies related to the alterations in the level of ceruloplasmin further support a dominant role of this protein, suggesting possible therapeutic applications. For example, international patent application No WO9825954 relates to the use of modified ceruloplasmin comprising a glycosylphosphatidylinositol moiety and its use for the treatment of toxic level of ferrous iron. Although the ROS scavenging capacities of CP has been shown in vitro, none of these studies has suggested the use of CP as neuroprotective agent neither they have shown that CP was sensitive in this action to the presence of selected antioxidants. These studies have not demonstrated or suggested either that CP could have a synergic neuroprotective action against oxidative stress when used in combination with a formulation of pyruvate, antioxidant and fatty acids. These discoveries were therefore unexpected and open the field of more powerful therapeutic applications to these known compounds.
In view of the above, it is clear that there is a need for neuroprotective compositions comprising ceruloplasmin (and/or derivatives thereof). There is also a need for antioxidative neuroprotective compositions comprising ceruloplasmin, pyruvate, antioxidant(s), lipid(s) such as fatty acid since these four components synergistically enhance their respective neuroprotective effects against oxidative stress, as it will be shown herein after.
The purpose of this invention is to fulfil these needs along with other needs that will be apparent to those skilled in the art upon reading the following specification.
The present invention relates to a neuroprotective composition and more particularly to a composition comprising ceruloplasmin and its uses.
According to an aspect of the invention, the neuroprotective composition comprises a therapeutically effective amount of ceruloplasmin or a functional derivative thereof. It is characterized in that it protects neuronal cells from reactive oxygen species such as xe2x80xa2O2xe2x88x92 and xe2x80xa2OH. Advantageously, ceruloplasmin or its functional derivative is purified from blood using an one-step affinity chromatography on aminoethyl-agarose.
According to another aspect of the invention, the neuroprotective composition further comprises an antioxidant consisting of: i) catalase; and/or ii) an amphiphilic physiological antioxidative solution, ceruloplasmin or the functional derivative of ceruloplasmin and the antioxidant being present in an amount that have a synergistic beneficial effect on neuronal cells. In a preferred embodiment, the amphiphilic physiological antioxidative solution comprises a mixture of pyruvate, at least one antioxidant and at least one lipid.
In a preferred embodiment, lipids consist of a mixture of saturated and unsaturated fatty acids selected from the group consisting of monogylcerides, digylcerides, trigylcerides, free fatty acids, and mixtures thereof.
Preferably, pyruvate is selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, prodrugs of pyruvic acid, and mixtures thereof.
Preferably, also the antioxidant is selected from lipid-soluble antioxidants, and more preferably the antioxidant is selected from the group consisting of Vitamin A, carotene, Vitamin E, pharmaceutically acceptable salts thereof, and mixtures thereof.
According to an other aspect of the invention, the neuroprotective composition is used as such or as an active agent in the preparation of a medication for the treatment of neuronal cells. Such treatments include the treatment brain trauma, brain or cerebrovascular ischemia, neurodegenerative diseases, poisoning of neuronal cells, the diminution of drugs side effects and for preservation of neuronal grafts.
According to an other aspect of the invention, the invention provides a method for treating neuronal oxidative stress related condition, the method comprising administrating to a patient in need thereof i) a therapeutically effective amount of ceruloplasmin or a functional derivative thereof; or ii) component i) in combination with catalase or in combination with a mixture of pyruvate, at least one antioxidant and at least one lipid.
Alternatively, the invention also provides a method for treating neuronal oxidative stress related condition comprising: a) administrating to a patient in need thereof, a therapeutically effective amount of an antioxidative composition comprising ceruloplasmin or a functional derivative thereof, pyruvate and at least one antioxidant; and b) providing, into the blood circulation of this patient, at least one lipid having a synergistic therapeutic effect on neuronal cells in combination with said antioxidative composition. The lipid(s) could be provided to the patient by increasing its lipidic blood level ratio through its diet. Examples of neuronal oxidative stress related condition include a neurodegenerative disease, such as amyotrophic lateral sclerosis, Alzheimer""s disease, Parkinson""s disease Huntington""s disease, etc, brain trauma, brain or cerebrovascular ischemia, neuronal cells poisoning, side effects caused by a drug and the preservation of neuronal grafts.
According to an other aspect of the invention it is provided a method for preparing a neuroprotective composition, the method comprising the steps of:
a) providing a therapeutically effective amount of i) ceruloplasmin or a functional derivative of ceruloplasmin, ii) pyruvate, iii) at least one antioxidant and iv) at least one lipid; and
b) mixing together components i), ii), iii) and iv) of step a) in a buffered saline solution to obtain a pharmaceutically acceptable homologous suspension; and optionally
c) centrifuging or filtering the homologous suspension obtained in step b).
The buffered saline solution may comprises sodium, potassium, magnesium and calcium ions at physiological concentrations and if necessary, an emulsifier.
An advantage of the present invention is that it provides effective means for maintaining or stimulating the capacity of neuronal cells to maintain or recover their viability or function in conditions of oxidative stress. It can also protect neuronal cells from a toxic substance or a stress, stabilizes the cellular membrane of a neuronal cell and/or helps in the normalization of neuronal cellular functions.
Other objects and advantages of the present invention will be apparent upon reading the following non-restrictive description of several preferred embodiments made with reference to the accompanying drawings.